Wild-type and transactivation-defective mutants of human immunodeficiency virus type 1 Tat protein bind human TATA-binding protein in vitro.

Tat regulates human immunodeficiency virus type 1 (HIV-1) gene expression by increasing both the rate of transcription initiation and the efficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that components of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with the human TATA-binding protein (TBP) and specific TBP-associated factors (TAFS) that comprise the TFIID complex. Here, in vitro glutathione S-transferase protein binding assays containing fully functional or transactivation-defective mutant Tat proteins have been used to investigate the functional significance of the direct interaction between Tat and TBP relative to Tat transactivation. Results demonstrate that full-length Tat, as well as the activation domain of Tat alone, binds human TBP in vitro. Site-directed mutations within the activation domain of Tat (C22G and P18IS) that abrogate transactivation by Tat in vivo fail to inhibit Tat-TBP binding. Full-length Tat, the activation domain of Tat alone, and a transactivation-defective mutant of Tat that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TBP provided that the H1 alpha helical domain that maps to amino acids 167-220 within the highly conserved carboxyl terminus of TBP is maintained. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding. is required for transactivation by Tat.